This invention relates to polymer-based microspheres, wherein the microspheres are labeled with one or more types of fluorescent nanocrystals. The resulting fluorescent microspheres are useful as distinct tagging agents or as encoders in methods utilizing fluorescence-based detection such as microarrays, high-throughput screening, immunoassays, and flow cytometry.
Fluorescent polymeric microspheres have been described as comprising either microspheres which are surface-labeled (including surface-coated) with fluorescent dyes, or microspheres having structurally incorporated within their volume (e.g., embedded or polymerized therein) a fluorescent dye. Commonly used fluorescence-based analyses that utilize fluorescent microspheres generally apply the microspheres for a purpose selected from the group consisting of: as a detection reagent with an affinity ligand bound thereto in assaying for the presence of a molecule for which the affinity ligand has binding specificity, as a calibrating agent for calibrating fluorescence-based detection systems, as a tracer (e.g., to trace the flow of a fluid containing the microspheres), and a combination thereof.
Typically, conventional fluorescent dyes (e.g., fluorescein, rhodamine, phycoerythrin, and the like) are used for labeling microspheres. These conventional fluorescent dyes typically have an excitation spectrum that may be quite narrow; hence, it is often difficult to find a wavelength spectrum of light suitable for simultaneously exciting several different fluorescent labels (e.g., differing in color of fluorescence emission). However, even when a single light source is used to provide a excitation wavelength spectrum (in view of the spectral line width), often there is insufficient spectral spacing between the emission optima of different species (e.g., differing in color) of fluorescent dyes to permit individual and quantitative detection without substantial spectral overlap. Additionally, conventional fluorescent dyes are susceptible to photobleaching which limits the time in which a fluorescent signal can be detected, and limits time-resolved fluorescence (fluorescent signal integration over time). Additional limitations of fluorescent dyes include fluorescence quenching, and shifts in fluorescence emission spectra, depending on the environment in which dyes are excited.
Fluorescent nanocrystals comprising either semiconductor nanocrystals or doped metal oxide nanocrystals have been reported to resist photobleaching, share an excitation wavelength spectrum, and are capable of emitting fluorescence of high quantum yield and with discrete peak emission spectra. However, these nanocrystals lack sufficient solubility in aqueous-based environments required for labeling microspheres; i.e., in aqueous-based environments, the nanocrystals interact together in forming aggregates, which leads to irreversible flocculation of the nanocrystals.
Thus, there remains a need for fluorescent microspheres that: (a) may be used in either single color or multicolor analysis; (b) are comprised of fluorescent nanocrystals which are sufficiently soluble in aqueous-based solutions to permit an effective concentration of the fluorescent nanocrystals to be embedded into polymeric microspheres in forming fluorescent microspheres; (c) which may be excited with a single wavelength spectrum of light resulting in detectable fluorescence of high quantum yield and with discrete peak emission spectra; (d) that are not susceptible to photobleaching and (e) which may further comprise one or more molecules of affinity ligand for use in fluorescence-based detection systems.
Provided is a fluorescent microsphere comprised of a polymeric microsphere that has embedded in its surface a plurality of at least one type of fluorescent nanocrystals, such that following excitation of the fluorescent microsphere with an appropriate excitation wavelength spectrum, the fluorescent microsphere will emit fluorescence of high quantum yield and with discrete peak emission. Fluorescent microspheres may differ in size and composition, and can be varied in the intensity of fluorescence emission and the one or more colors of fluorescence emission by changing the amount and type of fluorescent nanocrystals, respectively, in the method of preparing the microspheres. Thus, the fluorescent properties of the fluorescent microspheres, such as intensity and color, are sensitive to the corresponding fluorescent nanocrystals used to produce the fluorescent microspheres. A resultant advantage of the fluorescent microspheres of the present invention is that they may be used for measuring a plurality of analytes in a single sample. For example, a first population of fluorescent microspheres (a) may have embedded in their respective surface a plurality of one type of fluorescent nanocrystals which will emit a red fluorescent signal upon excitation, and (b) be tagged with affinity ligand having binding specificity for a first analyte. A second population of fluorescent microspheres (a) may have embedded in their respective surface a plurality of one type of fluorescent nanocrystals which will emit a yellow fluorescent signal upon excitation, and (b) be tagged with affinity ligand having binding specificity for a second analyte. Thus, because the first and second populations of fluorescent microspheres can be excited with a single wavelength spectrum of light resulting in detectable fluorescence of high quantum yield and with discrete peak emission spectra, and because the first and second populations of fluorescent microspheres can bind their respective analyte (via their affinity ligand), the two populations of fluorescent microspheres can be mixed within one sample in simultaneously measuring for the presence of the first and second analytes.
Advantages of the fluorescent microspheres over individual nanocrystals which have been functionalized to include an affinity ligand, include the following. First, a single nanocrystal may only be functionalized with a limited number of affinity ligands. In contrast, a fluorescent microsphere according to the present invention can have embedded in its surface a far greater number of fluorescent nanocrystals in enabling a fluorescent signal much greater in intensity than a single fluorescent nanocrystal can emit. Such an advantage is particularly useful in measuring an analyte which is present in minute quantities (and hence, can be bound by only a limited number of affinity ligand). Additionally, more than one type of fluorescent nanocrystal may be used for embedding into the surface of a fluorescent microsphere. Hence, the fluorescent microsphere may comprise a plurality of types of fluorescent nanocrystals embedded in its surface in encoding the fluorescent microsphere with a specific, identifiable code (based on the emission spectra which can comprise both color and intensity) which can be used to distinguish a population of these microspheres from a population of fluorescent microspheres which are encoded with a different fluorescence pattern. Such encoded fluorescent microspheres are particularly suited for multi-dimensional microarray formats. Further, in a method of producing the fluorescent microspheres according to the present invention, by controlling the proportion of starting materials (e.g., number of polymeric microspheres, and the number and composition of fluorescent nanocrystals), precise control may be achieved with respect to the basic fluorescent properties of the resultant fluorescent microspheres. Also provided is a kit comprising the fluorescent microspheres, and may further comprise the fluorescent microspheres operably bound to affinity ligand.
The above and other objects, features, and advantages of the present invention will be apparent in the following Detailed Description of the Invention when read in conjunction with the accompanying drawings.